The objective of this research program is to investigate the relationships between the biochemical structures of heparins and their anticoagulant activities. The basis for the study is the investigation of heparins fractionated by a new separatory technique. The objective of fractionation is to separate heparins into chemically more defined and homogeneous fractions which vary systematically in structural or compositional parameters. Different heparins, including clinical heparins, purified hog mucosal and beef lung heparins, mast cell heparins, and antithrombin-affinity fractionated heparins will be examined. In vitro anticoagulant activities of fractions will be measured with the USP, antithrombin inhibition of thrombin (thrombin inactivation), or factor Xa (anti-Xa), and protamine neutralization assays. Objectives are: (1) to determine the relationship between these assays and the structure of the heparin; (1) to provide the basis for better comparison of different in vitro measurements; (3) to compare properties and potencies of different and heterogeneous heparins in terms of their constituents, and (4) to determine the possibilities for improved standardization of heparin assays. In order to further extend this approach, the analytical chemistry of heparin will be investigated by: (1) investigating behavior of heparins in different two-phase solutions to identify other structural parameters as the bases for separations; (2) further development of countercurrent chromatography for obtaining continuous flow, high resolution preparative and analytical separations of heparins, and (3) explore the use of optical rotation as the the basis for an on-line chromatographic detector capable of qualitative and quantitative measurements. In order to obtain more definitive structural information with which to relate to anticoagulant activities of the heparin fractions, the final objectives will be to develop methylation analysis coupled with GC/MS to identify and measure various constituents of the heparin molecule.